[methods in enzymology] chromatin and chromatin remodeling enzymes, part c volume 377 || genetic and...

to a greater extent than they compromise abd-A and/or Abd-B expression).A simplified version of this model to account for the ETP group effects isshown in Fig. 3. I am currently trying to test this model by independentlyaltering expressions of the abd-A and Abd-B genes in the genetic assaysthat identified the ETP group. If true, the ETP group should remain asubset of the Pc-G.

Fig. 3. Model for homeotic cross-regulatory interactions and the ETP group assay. At the

top are shown the Abd-B (repressed) and Ubx (expressed) genes in the wild-type third thoracic

segment. In the lower half are shown the effects of partial derepression of Abd-B by an ETP

group mutation and partial loss of Ubx function by a Trx-G mutation. The ectopic Abd-B protein

(shown as a cross-hatched circle) binds to sequences in the Ubx gene and represses Ubx

transcription, further enhancing the loss of function caused by the Trx-G mutation.

70 chromatin modification and remodeling [4]

[4] Genetic and Cytological Analysis of DrosophilaChromatin-Remodeling Factors

By Davide F. V. Corona, Jennifer A. Armstrong, andJohn W. Tamkun

Eukaryotic DNA is packaged into a highly compact, dynamic structurecalled chromatin. Although chromatin provides the cell with the obviousbenefit of organizing a large and complex genome, it can also blockthe access of transcription factors and other proteins to DNA.1 Twoclasses of enzymes, ATP-dependent chromatin-remodeling factors and

Copyright 2004, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 377 0076-6879/04 $35.00

[4] IN VIVO analysis of DROSOPHILA chromatin-remodeling factors 71

histone-modifying enzymes, modulate chromatin accessibility by directlymodifying the structure and/or position of nucleosomes.2–4 These enzymesfunction in a variety of nuclear processes including gene expression.5,6 Bothclasses of enzymes typically function in the context of multi-subunitcomplexes. Current efforts are directed towards understanding how theactivities of these complexes are targeted, regulated, and coordinatedin vivo.7,8

Drosophila melanogaster is a particularly useful model system for thestudy of chromatin remodeling and modifying enzymes since it permitsboth the genetic and biochemical analysis of these factors in a developingorganism. We use Drosophila to examine the biological roles of severalchromatin-remodeling factors, including Brahma (BRM) and Imitation-SWI (ISWI). brm was identified as a suppressor of Polycomb9 and encodesthe ATPase subunit of the BRM chromatin-remodeling complex: a 2-MDacomplex highly related to the S. cerevisiae SWI/SNF and RSC com-plexes.10–12 BRM maintains the transcription of homeotic genes duringDrosophila development13 and functions as a global activator of transcrip-tion.14 The ISWI ATPase functions as the catalytic subunit of three distinctchromatin-remodeling complexes, NURF, CHRAC, and ACF, which cata-lyze nucleosome remodeling and spacing reactions in vitro.15 ISWI hasbeen implicated in both transcriptional activation and repression16,17 aswell as the maintenance of higher order chromatin structure.16,18

1 G. Felsenfeld and M. Groudine, Nature 421, 448 (2003).2 T. Jenuwein and C. D. Allis, Science 293, 1074 (2001).3 P. B. Becker and W. Horz, Ann. Rev. Biochem. 71, 247 (2002).4 B. M. Turner, Cell 111, 285 (2002).5 J. A. Martens and F. Winston, Curr. Opin. Genet. Dev. 13, 136 (2003).6 M. Iizuka and M. M. Smith, Curr. Opin. Genet. Dev. 13, 154 (2003).7 S. L. Berger, Curr. Opin. Genet. Dev. 12, 142 (2002).8 G. J. Narlikar, H. Y. Fan, and R. E. Kingston, Cell 108, 475 (2002).9 J. A. Kennison and J. W. Tamkun, Proc. Natl. Acad. Sci. USA 85, 8136 (1988).

10 O. Papoulas, S. J. Beek, S. L. Moseley, C. M. McCallum, M. Sarte, A. Shearn, and J. W.

Tamkun, Development 125, 3955 (1998).11 R. T. Collins, T. Furukawa, N. Tanese, and J. E. Treisman, EMBO J. 18, 7029 (1999).12 A. J. Kal, T. Mahmoudi, N. B. Zak, and C. P. Verrijzer, Genes Dev. 14, 1058 (2000).13 J. A. Simon and J. W. Tamkun, Curr. Opin. Genet. Dev. 12, 210 (2002).14 J. A. Armstrong, O. Papoulas, G. Daubresse, A. S. Sperling, J. T. Lis, M. P. Scott, and J. W.

Tamkun, EMBO J. 21, 5245 (2002).15 G. Langst and P. B. Becker, J. Cell Sci. 114, 2561 (2001).16 R. Deuring, L. Fanti, J. A. Armstrong, M. Sarte, O. Papoulas, M. Prestel, G. Daubresse,

M. Verardo, S. L. Moseley, M. Berloco, T. Tsukiyama, C. Wu, S. Pimpinelli, and J. W.

Tamkun, Mol. Cell 5, 355 (2000).17 P. Badenhorst, M. Voas, I. Rebay, and C. Wu, Genes Dev. 16, 3186 (2002).18 D. F. Corona, C. R. Clapier, P. B. Becker, and J. W. Tamkun, EMBO Rep. 3, 242 (2002).


In this chapter, we present methods that have proven useful in our stud-ies of BRM and ISWI. We describe strategies for the generation and analy-sis of dominant-negative mutations in chromatin-remodeling factors andtheir use in genetic modifier screens. We also discuss the use of polytenechromosomes to study interactions between chromatin-remodeling factorsand chromatin in vivo.

Use of Dominant-Negative Mutations for Studying EnzymesInvolved in Chromatin-Remodeling

Due to their critical roles in gene expression and development, manyDrosophila chromatin-remodeling factors are encoded by essential genes.Mutations in these genes can block oogenesis and cell proliferation16,19,20;as a result, it is difficult to generate cells or organisms that completely lacktheir activity. This problem can be circumvented using either conditional ordominant-negative alleles. Due to the ease with which they can be gener-ated, dominant-negative mutations are particularly useful for studying thefunction of chromatin-remodeling factors. Replacing a conserved lysine inthe ATP-binding site of a chromatin-remodeling factor with an arginineeliminates its catalytic activity without disrupting its ability to interact withother proteins.20–24 These mutant proteins therefore have strong, domi-nant-negative effects when expressed at high levels in vivo. Similar muta-tions can be used to study the function of virtually any complex withchromatin-remodeling or modifying activity.

Generation and Characterization of Dominant-Negative Transgenes

A variety of inducible or tissue-specific promoters can be used to drivethe expression of cDNA or genomic DNA fragments encoding dominant-negative chromatin-remodeling factors. Ideally, it is best to begin by ex-pressing the dominant-negative transgene under the control of its normalpromoter to ensure that it is expressed in a spatial and temporal pattern

19 M. L. Ruhf, A. Braun, O. Papoulas, J. W. Tamkun, N. Randsholt, and M. Meister,

Development 128, 1429 (2001).20 L. K. Elfring, C. Daniel, O. Papoulas, R. Deuring, M. Sarte, S. Moseley, S. J. Beek, W. R.

Waldrip, G. Daubresse, A. DePace, J. A. Kennison, and J. W. Tamkun, Genetics 148, 251

(1998).21 E. Richmond and C. L. Peterson, Nucleic Acids Res. 24, 3685 (1996).22 P. A. Khavari, C. L. Peterson, J. W. Tamkun, D. B. Mendel, and G. R. Crabtree, Nature

366, 170 (1993).23 J. Cote, J. Quinn, J. L. Workman, and C. L. Peterson, Science 265, 53 (1994).24 D. F. Corona, G. Langst, C. R. Clapier, E. J. Bonte, S. Ferrari, J. W. Tamkun, and P. B.

Becker, Mol. Cell 3, 239 (1999).


comparable to that of its wild-type counterpart. After transgenic Drosoph-ila strains are generated, the ability of the transgene to rescue null muta-tions should be tested to verify that the mutation destroys the activity ofthe chromatin-remodeling factor. Western blotting and gel filtration chro-matography should be used to ensure that the mutant protein is expressedat normal levels and incorporated into chromatin-remodeling complexes.We routinely place an epitope tag at the N- or C-terminus of the mutantprotein so that it can be distinguished from the endogenous protein by im-munofluorescence or Western blotting. Sufficient material for these bio-chemical assays can be obtained from relatively small numbers ofembryos using the following protocol.

Preparation of Protein Extracts From Small Quantitiesof Drosophila Embryos

1. Place approximately 25 male and 25 female adults on grape juice-agar plates (2.25% agar, 0.625% sucrose, 25% concentrated grapejuice, 0.015% methyl paraben) streaked with yeast paste. Changethe plates every 12 h until the females begin laying sufficientnumbers of eggs (approximately 2 days).

2. Collect 0–12 h embryos on grape juice-agar plates. One collectionroutinely yields about 50 to 100 �l of embryos. If necessary, embryoscan usually be stored at 4

�for 2 days before extracts are prepared.

3. Collect embryos into a small Nitex mesh sieve while rinsing withdeionized water. Dechorionate embryos by incubation for 2 min in afreshly made solution of 50% bleach (2.6% sodium hypochlorite)with occasional gentle swirling.

4. Rinse embryos three times in deionized water and once in washbuffer (0.4% NaCl, 0.03% Triton X-100) for 1 min to removebleach.

5. Gently scoop dechorionated embryos into 2 ml dounce homoge-nizer. Cover the embryos with wash buffer and let them settle on icefor 3 min.

6. Aspirate off the wash buffer and floating chorions. Remove excessliquid by placing the tip of a Pasteur pipette against the bottom ofthe dounce; capillary action will remove most of the excess liquid.

7. Add an equal volume of homogenization buffer (50 mM HEPES pH7.6, 385 mM NaCl, 0.1% Tween, 0.1 mM EGTA, 1 mM MgCl2, 10%glycerol, 1� complete protease inhibitors [Roche #1697498]) to thepacked embryos. Homogenize the embryos with 10 strokes of a tightpestle. Let the homogenate sit on ice for 10 min to extract proteinsfrom the damaged nuclei.


8. Centrifuge the homogenate at 4�

for 30 min at 25,000 rpm in aTLA100.2 rotor. Remove the top lipid layer by touching it with thewide end of a 200 �l plastic pipette tip layer and quickly pulling it up.

9. Transfer the supernatant to an Eppendorf tube and determine itsvolume and protein concentration by standard Bradford assay.

The above protocol routinely yields 200 �l of extract with a proteinconcentration of 10–30 �g/�l. The extract can be used immediately forWestern blotting, gel filtration chromatography, immunoprecipitationor other assays. Alternatively, the extract can be flash frozen in liquidnitrogen and stored at �80

�for later use.

Functional Characterization of Chromatin-Remodeling Factors UsingDominant-Negative Transgenes

Information concerning the function of chromatin-remodeling factorsin vivo can often be obtained by characterizing phenotypes resulting fromthe expression of dominant-negative transgenes. For example, the expres-sion of a dominant-negative form of the BRM protein (BRMK804R)revealed an unanticipated role for this chromatin-remodeling factor in pe-ripheral nervous system development.20 The GAL4 system of Brand andPerrimon25 is particularly useful for this purpose because it allows highlevels of a dominant-negative transgene to be expressed in a wide varietyof different cell or tissue types. To use the GAL4 system, a cDNA or geno-mic DNA fragment encoding a dominant-negative chromatin-remodelingfactor is placed downstream of a minimal promoter and GAL4-responsiveelement in the transformation vector pUAST.25 To induce expression ofthe dominant-negative protein, a strain bearing the transgene is crossedto a strain bearing a ‘‘driver’’ gene that expresses GAL4 in a temporallyor spatially restricted pattern.25–27 A current list of available driver linesand their expression patterns may be obtained from the Bloomington StockCenter (http://flystocks.bio.indiana.edu/gal4.htm).

Eye-based Screens for Identifying Genes that Interact withChromatin-Remodeling Factors In vivo

Although in vitro systems have provided a wealth of information con-cerning the mechanism of action of chromatin-remodeling factors, theydo not reflect the complexity of chromatin that exists in vivo. Furthermore,

25 A. H. Brand and N. Perrimon, Development 118, 401 (1993).26 P. P. D’Avino and C. S. Thummel, Methods Enzymol. 306, 129 (1999).27 J. B. Duffy, Genesis 34, 1 (2002).

http://flystocks.bio.indiana.edu/gal4.htm


proteins that directly or indirectly regulate the activity of chromatin-remodeling factors can be difficult to detect using biochemical assays. Bycontrast, genetic screens have the potential to identify novel compo-nents of chromatin and other proteins that functionally interact withchromatin-remodeling factors without bias concerning their mechanismof action.

The Drosophila eye provides an attractive system for the genetic analy-sis of complex biological processes, including chromatin-remodeling. Sincethe eye is not required for viability, it is relatively straightforward to studythe function of essential genes in this tissue. Screens for modifiers ofphenotypes resulting from the expression of wild-type or dominant-negative proteins in the developing eye have been used to analyze a widevariety of regulatory pathways.28

Phenotypes resulting from the expression of dominant-negative chro-matin-remodeling factors using the GAL4 system are ideal for eye-basedmodifier screens. Many different GAL4 drivers can be used to drive the ex-pression of dominant-negative transgenes in the eye-antennal disc. We rou-tinely use a GAL4 driver under the control of the eyeless regulatoryelement (ey-GAL4).29 The expression of either BRMK804R (see Fig. 1B)or ISWIK159R in the eye-antennal disc leads to the development of adultswith rough, reduced, or missing eyes16; this phenotype can be quantita-tively scored for enhancement or suppression, allowing the identificationof genes that interact with chromatin-remodeling factors in vivo. Beforeinitiating a dominant-modifier screen, it is important to verify that theeye defects result from the loss of function of the chromatin-remodelingfactor of interest. For example, the rough eye phenotype resulting from ex-pression of BRMK804R is enhanced by reducing the level of wild-type BRM(see Fig. 1C). The sensitivity of the assay should be tested using mutationsin candidate genes. For example, mutations in a gene encoding a subunit ofthe BRM complex, Moira (MOR),30 strongly enhance eye defects resultingfrom the expression of BRMK804R (see Fig. 2). To gain confidence in theselectivity of the screen, it is important to show that mutations in function-ally unrelated chromatin-remodeling factors fail to interact in the eyeassay. For example, ISWI mutations have no effect on eye defects resultingfrom BRMK804R expression.

Eye-based assays can be used to investigate functional interactionsbetween chromatin-remodeling factors and other proteins known to

28 B. J. Thomas and D. A. Wassarman, Trends Genet. 15, 184 (1999).29 D. J. Hazelett, M. Bourouis, U. Walldorf, and J. E. Treisman, Development 125, 3741 (1998).30 M. A. Crosby, C. Miller, T. Alon, K. L. Watson, C. P. Verrijzer, R. Goldman-Levi, and

N. B. Zak, Mol. Cell. Biol. 19, 1159 (1999).

Fig. 1. Expression of dominant-negative brm (brmK804R) in the developing eye results in a

rough eye phenotype. (A) Scanning electron micrograph of wild-type eye. (B) Eye from an

individual expressing brmK804R under control of the ey-GAL4 driver. To facilitate the eye-

based assay, the ey-GAL4 transgene was recombined onto the chromosome carrying the

UAS-brmK804R transgene. (C) A null mutation in the brm gene (brm2) enhances the rough eye

phenotype of ey-GAL4, UAS-brmK804R.


modulate chromatin structure or gene expression. For example, using aneye-based assay we were able to demonstrate a functional antagonism be-tween ISWI and the MOF histone acetyltransferase in vivo.18 We were alsoable to confirm that the HMG-domain protein BAP111 is critical for thefunction of the BRM complex.31

Eye-based screens can also be used to identify potentially novel genesthat interact with chromatin-remodeling factors. The GAL4 system is

31 O. Papoulas, G. Daubresse, J. A. Armstrong, J. Jin, M. P. Scott, and J. W. Tamkun, Proc.

Natl. Acad. Sci. USA 98, 5728 (2001).

Fig. 2. A mor allele (mor6) enhances eye defects resulting from the expression of

brmK804R. ey-GAL4, UAS-brmK804R/TM3, Sb males were crossed to mor6/TM6B, Hu virgins.

Eyes of the progeny were scored using an arbitrary scale of 1 to 6: (1) normal eye; (2) 50% or

less of the eye is rough (as determined by disordered ommatidia under the light microscope);

(3) greater than 50% of the eye is rough; (4) the eye is rough and reduced in size by 50% or

less; (5) the eye is rough and reduced in size by more than 50%; and (6) eye is absent. We use

the Kolmogorov–Smirnov two-sample test to determine the statistical significance of the

genetic interactions.


inherently temperature sensitive25; progeny with moderate eye defects canbe generated by varying the temperature between 18

�and 29

�, thus

allowing the recovery of both suppressor and enhancer mutations in anF1 screen. An F2 screen should be considered, however, if the number ofprogeny that display extremely strong or weak phenotypes is too high(>0.1%). Examples of both F1 and F2 screens for dominant modifiers ofeye phenotypes are shown in Fig. 3. Screens for EMS-induced mutationshave strong tendency to recover loss-of-function mutations. As a result,mutations in genes that are not expressed in limiting quantities may notbe recovered in dominant-modifier screens. An alternative approach is toscreen for genes that modify eye phenotypes when expressed at high levels.This approach has been greatly facilitated by the availability of a large

Fig. 3. Schemes for F1 and F2 eye-based modifier screens. In an F1 screen, males are

mutagenized and crossed en masse to virgins expressing the dominant-negative chromatin-

remodeling factor in the eye. Single F1 males are scored for enhancement or suppression of

eye defects. Candidate modifier mutations are retested and stocks are established. In an F2

screen, single F1 progeny bearing mutagenized chromosomes are crossed to virgin females

expressing the dominant-negative chromatin-remodeling factor in the eye. F2 progeny are

scored for enhancement or suppression of eye defects. Siblings carrying the mutation over

a balancer are used to establish stocks and retest. Protocols for F1 and F2 screens for

EMS-induced mutations have been published elsewhere.32


collection of inducible P insertions known as EPs (http://flystation.exelixis.com/).33,34 Each insertion contains a GAL4-responsive promoter thathas the potential to drive high levels of expression of a neighboring gene.These genes can be easily identified by their annotation in the BerkeleyDrosophila Genome Project (http://www.fruitfly.org/index.html).

Once candidate mutations are identified, other criteria should be usedto identify the most promising mutations for further analysis. For example,to test for specificity we ask whether a mutation that modifies a brmK804R

rough eye phenotype has any affect on an ISWIK159R rough eye phenotype,and vice versa. This allows us to exclude mutations that modify dominant-negative phenotypes for trivial reasons, such as altering the expression ofthe UAS transgenes. Standard techniques can then be used to map andcharacterize the genes identified in the modifier screens. Ultimately, the

32 T. Grigliatti, in ‘‘Drosophila a Practical Approach’’ (D. B. Roberts, ed.), p. 39. IRL Press,

Oxford, 1986.33 P. Rorth, K. Szabo, A. Bailey, T. Laverty, J. Rehm, G. M. Rubin, K. Weigmann, M. Milan,

V. Benes, W. Ansorge, and S. M. Cohen, Development 125, 1049 (1998).34 P. Rorth, Proc. Natl. Acad. Sci. USA 93, 12418 (1996).

http://flystation.exelixis.com/

http://www.fruitfly.org/index.html

http://flystation.exelixis.com/


biochemical characterization of the genes products will be necessary toclarify the basis of their genetic interactions with chromatin-remodelingfactors.

Using Salivary Gland Polytene Chromosomes to StudyChromatin-Remodeling Factors

One advantage of using Drosophila as a model system is the ability todirectly visualize interphase chromosomes. The chromosomes of the sali-vary gland and many other larval tissues undergo multiple rounds ofDNA replication in the absence of cell division. The resulting giantchromosomes display reproducible banding patterns that are easily visual-ized under the light microscope. Salivary gland polytene chromosomes canbe stained with antibodies against chromatin-remodeling factors and otherproteins to directly visualize their interactions with chromatin. Althoughthis approach lacks the resolution of chromatin immunoprecipitation(ChIP), it provides a straightforward and inexpensive way to determinethe genome-wide distribution of a protein of interest.

Many protocols for immunostaining polytene chromosomes have beenpublished.35–38 We have successfully used the following protocol to com-pare the distribution of several different proteins, including BRM, ISWI,the initiating and elongating forms of RNA polymerase II, and Polycomb.

Antibody Staining of Polytene Chromosomes

1. Dissect salivary glands of third-instar larvae in 0.7% NaCl. Removeas much of the fat as possible without damaging the glands.Detailed instructions on how to dissect and manipulate salivaryglands have been published elsewhere.35,39

2. Transfer one set of glands to 12 �l of freshly prepared fixingsolution (45% glacial acetic acid, 1.85% formaldehyde) on asilanized coverslip and fix for 10 min. To silanize coverslips, dipthem in 1% dimethylsilane in chloroform (work in a hood), and letdry overnight. Silanized coverslips can be stored indefinitely in adust free box.

35 L. A. Pile and D. A. Wassarman, Methods 26, 3 (2002).36 L. M. Silver and S. C. Elgin, Proc. Natl. Acad. Sci. USA 73, 423 (1976).37 R. Paro, in ‘‘Drosophila Protocols’’ (W. Sullivan, M. Ashburner, and R. S. Hawley, eds.),

p. 131. Cold Spring Harbor Press, Cold Spring Harbor, NY, 2000.38 D. J. Andrew and M. P. Scott, Methods Cell Biol. 44, 353 (1994).39 J. A. Kennison, in ‘‘Drosophila Protocols’’ (W. Sullivan, M. Ashburner, and R. S. Hawley,

eds.), p. 111. Cold Spring Harbor Press, Cold Spring Harbor, NY, 2000.


3. Pick up the coverslip by gently touching the drop of fixative with aclean slide. Place the slide (with the coverslip facing up) on a papertowel. Use the back end of a small paintbrush to disrupt the nucleiand spread the chromosomes. Start tapping in the center of thecoverslip and gently tap out in a spiral, repeat once. Bracket thecoverslip with gloved fingertips to keep it from sliding.

4. Being careful not to move the coverslip, cover the slide with apaper towel and squash with thumb. Check the quality of thechromosome squash by phase-contrast microscopy. A good squashwill have well-spread chromosome arms and distinct bandingpatterns.

5. Mark the position of the coverslip on the slide with a diamond pen.Freeze slide in liquid nitrogen and remove the coverslip by wedginga razor blade under the corner and flipping off the coverslip.Immediately place slide in PBS (137 mM NaCl, 2.68 mM KCl,10.14 mM Na2HPO4, 1.76 mM KH2PO4, adjust pH to 7.2 with 1 NNaOH). You can freeze slides as you go and accumulate them inPBS, otherwise they tend to dry out.

6. Wash slides in PBS for 5 min and in PBS-T (PBS containing 1%Triton X-100) for 10 min. Block slides in PBS-TB (PBS containing1% BSA and 0.1% Triton X-100) for 30 min.

7. Dilute primary antibody in PBS-TB (when diluting antibodies, useRIA grade BSA, Sigma). In general, the concentration of primaryantibody should be 10-fold higher than used for western blotting.Place 20 �l diluted primary antibody on a coverslip. Dry off asmuch of the slide as possible without disturbing the area containingthe chromosome squash. Pick up the coverslip with the slide,avoiding bubbles. Place slide with the coverslip facing up in ahumid chamber (a large Petri dish lined with wet kimwipes workswell for this purpose). Incubate overnight at 4

�.

8. Rinse slides in PBS to remove coverslips. Wash slides three timesfor 5 min each in PBS and twice for 15 min each in PBS-TB.

9. Dilute secondary antibody in PBS-TB according to the manufac-turer’s recommendations. For best results, the titer of thesecondary antibody should be determined empirically. Place 20 �ldiluted secondary antibody on a coverslip. Dry the slide and pickup the coverslip as before. Place in a humid chamber in the dark atroom temperature for 1 h.

10. Rinse slides in PBS to remove the coverslips and wash three timesfor 5 min each in PBS.

11. If desired, place 20 �l of 0.05 �g/ml DAPI dissolved in 2� SSC(0.3 M NaCl, 0.03 M sodium citrate, 0.1 mM EDTA) on a coverslip


and pick up with the slide as before. Incubate 4 min at roomtemperature.

12. Wash slides three times for 5 min each in PBS. Place slides on 20 �lof PBS containing 80% glycerol and 2.5% n-propylgallate on acoverslip. Place slides face down on paper towels and gently presswith fingers to remove excess mountant. Seal with nail polish ifdesired. Slides can be stored lying flat in the dark at 4

�.

13. Visualize with a fluorescent microscope using appropriate filters.

Several controls can be used to confirm that the results obtained usingthe above protocol accurately reflect the chromosomal distribution of theprotein of interest. Whenever possible, affinity-purified or monoclonal anti-bodies should be used and the specificity of the primary antibody should beconfirmed by Western blotting. The specificity of the secondary antibodyshould be confirmed by omitting the primary antibody. These controls arecritical because the quality of the results obtained using the above protocolis highly dependent on the quality of the primary and secondary antibodies.If possible, one should compare results obtained using antibodies againstdifferent epitopes of the same protein, or different subunits of the samecomplex. The ideal control is to show that the staining pattern is reducedor eliminated in larvae homozygous for a null allele of the gene of interest.However, this is not feasible for most chromatin-associated proteins,including those that are essential for early development.

Staining Polytene Chromosomes with Two Antibodies

The distribution of a chromatin-remodeling factor relative to RNApolymerase II (Pol II) and other proteins can be quite informative. Forexample, the chromosomal distribution of a chromatin-remodeling factorthat plays a general role in facilitating transcription might be similar to thatof Pol II, as has been observed for the BRM complex.14 Antibodies specificto the paused, hypophosphorylated form of RNA Pol II, as well as the hy-perphosphorylated, elongating forms of Pol II can be used to clarifywhether a protein of interest facilitates a specific step of transcription.The comparison of chromatin-associated factors to specific forms of modi-fied histones can also provide insights concerning their interactionsin vivo.40,41 Note that antibodies directed against modified histones (andother basic proteins) require specific staining protocols.16,42,43

40 L. A. Pile and D. A. Wassarman, EMBO J. 19, 6131 (2000).41 S. A. Jacobs, S. D. Taverna, Y. Zhang, S. D. Briggs, J. Li, J. C. Eissenberg, C. D. Allis, and

S. Khorasanizadeh, EMBO J. 20, 5232 (2001).


Assuming that the necessary immunological reagents are available, it isrelatively straightforward to stain chromosomes with two or more anti-bodies. Polytene chromosomes can be stained with a mixture of primaryantibodies raised in different species, followed by an appropriate combi-nation of species-specific secondary antibodies. As controls, each of the pri-mary antibodies must be omitted in turn to ensure that the secondaryantibodies do not cross react with inappropriate primary antibodies.

Other approaches must be used in situations where the primary anti-bodies were generated in the same species. Primary antibodies can be cova-lently coupled to fluorochromes, thereby eliminating the need forsecondary antibodies.44 We have used this approach to compare the distri-butions of ISWI and GAGA factor on salivary gland polytene chromo-somes.16 One limitation of direct labeling is that it requires relativelylarge amounts of primary antibody. This technique often yields suboptimalresults due to the loss of signal amplification resulting from the omissionof secondary antibodies. Direct labeling is therefore best suited to robustantibodies against relatively abundant proteins.

Another technique utilizes Fab fragments to allow the comparison ofthe chromosomal distributions of two proteins, even when the relevant pri-mary antibodies were raised in the same species and are present in limitedquantities. In this technique, Fab fragments are used to both label andblock the first primary antibody. We have used the following protocol(adapted from a method suggested by Jackson ImmunoResearch Labora-tories, West Grove, PA) to stain chromosomes with rabbit antibodiesdirected against BRM and Polycomb.14

Using Fab Fragments to Stain Polytene Chromosomes with Two PrimaryAntibodies Raised in Rabbits

1. Prepare, wash, and block squashes of salivary gland chromosomes asdescribed earlier.

2. Dilute first primary antibody with PBS-TB and place on slide asdescribed earlier. It is best to choose the antibody with the fewestbinding sites to be the first primary antibody. Incubate at roomtemperature for 1 h.

42 J. Fang, Q. Feng, C. S. Ketel, H. Wang, R. Cao, L. Xia, H. Erdjument-Bromage, P. Tempst,

J. A. Simon, and Y. Zhang, Curr. Biol. 12, 1086 (2002).43 K. Nishioka, J. C. Rice, K. Sarma, H. Erdjument-Bromage, J. Werner, Y. Wang, S. Chuikov,

P. Valenzuela, P. Tempst, R. Steward, J. T. Lis, C. D. Allis, and D. Reinberg, Mol. Cell 9,1201 (2002).

44 E. Harlow and D. Lane, in ‘‘Antibodies a Laboratory Manual,’’ p. 319. Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY, 1998.


3. Wash slides three times for 5 min each in PBS, two times for 15 mineach in PBS-TB. Add biotin-labeled goat antirabbit Fab fragments(Jackson ImmunoResearch Laboratories, West Grove, PA) diluted1:200 in PBS-TB for 1 h at room temperature. This step is necessaryto both label the primary antibody and to block the binding of otherantibodies to the primary antibody during subsequent steps.

4. Wash slides twice for 10 min each in PBS-TB. Incubate withfluorescently labeled streptavidin (Jackson ImmunoResearch Lab-oratories, West Grove, PA) diluted 1:400 in PBS-TB for 1 h at roomtemperature.

5. Wash slides twice for 10 min each in PBS-TB and stain with thesecond primary antibody and secondary antibody as describedearlier.

The second secondary antibody is blocked from binding the first pri-mary antibody by the Fab fragments. Therefore, this protocol will yieldspurious results if the Fab fragments fail to completely mask all bindingsites on the first primary antibody. As a control, the second primary anti-body should be omitted to verify that the fluorescently labeled secondaryantibody is unable to bind the first primary antibody following Fabblocking. If needed, unlabeled Fab fragments (Jackson ImmunoResearchLaboratories) can be used to further block the first primary antibody.Following incubation with the biotin-labeled Fab fragments, incubateslides with 20 �g/ml Fab fragments for 1 h at room temperature.

When directly comparing the distribution of two proteins in mergedimages of polytene chromosomes, high levels of one protein can masklow levels of another. As a result, the staining patterns of proteins with vir-tually identical distributions may appear quite different. This problem canbe circumvented by generating ‘‘split’’ images of polytene chromosomesusing Adobe Photoshop software as shown in Fig. 4.

Analysis of Chromosome Defects Resulting from the Loss ofChromatin-Remodeling Factor Function

The analysis of polytene chromosomes has revealed unanticipated rolesfor chromatin-remodeling factors in vivo. For example, the global architec-ture of the male X chromosome is dramatically altered in ISWI mutantlarvae, suggesting that this chromatin-remodeling factor is required for themaintenance of higher order chromatin structure.16 It is not always possibleto examine polytene chromosomes of homozygous mutant larvae, sincemany chromatin-remodeling factors are essential for early development.The expression of dominant-negative forms of chromatin-remodelingfactors can be used to circumvent this problem. For example, the expression

Fig. 4. ‘‘Split’’ chromosomes reveal that BRM and RNA Pol II colocalize on polytene

chromosomes. Salivary glands from wild type larvae were stained with rabbit anti-BRM and

goat anti-RNA Pol II (subunit IIc).45 To generate a ‘‘split’’ image in Photoshop, individual

images (BRM and IIc) were put in separate layers. The ‘‘lasso tool’’ was used to select half the

chromosome arm in the upper layer and delete the selection to reveal the underlying layer.

Displaying multiple staining patterns in a ‘‘split’’ format helps avoiding visual artifacts when

high levels of one protein can mask low levels of another.


of dominant-negative BRM protein in salivary gland nuclei decreases thelevels of both paused and elongating forms of Pol II associated with salivarygland chromosomes, suggesting that the BRM complex plays a global role ingene expression.14 To express dominant-negative proteins in salivary glandnuclei, we routinely use either the ey-GAL4 driver or the heat shock driverP[wþmC ¼ GAL4-Hsp70.PB]89-2-1, grown at 18

�under nonheat shock con-

ditions. When doing these types of experiments, it is critical to raise the fliesat constant temperature in uncrowded conditions. UAS-LACZ or UAS-GFP lines can be used as controls. The control and experimental chromo-somes must always be stained in parallel, and the resulting images must betaken with the same camera settings.

45 A. M. Skantar and A. L. Greenleaf, Gene Expr. 5, 49 (1995).

[5] genetic analysis of mammalian H1 85

Although this chapter focuses on methods we have used to study theBRM and ISWI chromatin-remodeling factors, similar approaches can beused to study other chromatin-remodeling and modifying enzymes. To-gether with biochemical studies, these genetic and cytological methodsshould facilitate the investigation of the complex regulatory network ofchromatin-modulating proteins.

Acknowledgments

We would like to thank Mary Kay Phillips from Jackson ImmunoResearch Laboratories

(West Grove, PA), for advice concerning the Fab fragment-based polytene staining protocol.

We also would like to thank Dr. Renate Deuring and Vidhya Srinivasan for their useful

comments on this manuscript. Most of the stocks described in this article can be obtained from

the Bloomington Stock Center or our laboratory. Work in our laboratory is supported by a

Grant from the NIH (GM49883) to J. W. T. J. A. A. has been supported by the Damon

Runyon Cancer Research Fund (Fellowship DRG-1556) and D. F. V. C. has been supported

by EMBO and HFSP postdoctoral fellowships.

[5] Genetic Analysis of H1 Linker Histone Subtypesand Their Functions in Mice

By Yuhong Fan and Arthur I. Skoultchi

In most eukaryotic cells, the chromatin fiber consists of nearly one mol-ecule of linker histone for each nucleosome core particle. Therefore, linkerhistones are expected to play a key role in the structure of the chromatinfiber. A large variety of in vitro experiments with chromatin, and other cor-relative findings, support this view. These studies indicate that two impor-tant functions of linker histones are to stabilize the DNA as it enters andexits the core particle and to facilitate the folding of nucleosome arrays intomore compact structures. Linker histones also affect nucleosome coreparticle spacing and mobility in vitro.1,2

Surprisingly, however, elimination of the linker histone in Tetrahymena,yeast and fungi led to the conclusion that H1 is not essential in these unicel-lular eukaryotes.3–7 But double stranded RNA-mediated interference

1 K. E. van Holde, ‘‘Chromatin.’’ Springer-Verlag, New York, 1989.2 A. P. Wolffe, ‘‘Chromatin: Structure and Function.’’ Academic Press, San Diego, CA, 1998.3 X. Shen, L. Yu, J. W. Weir, and M. A. Gorovsky, Cell 82, 47 (1995).4 S. C. Ushinsky, H. Bussey, A. A. Ahmed, Y. Wang, J. Friesen, B. A. Williams, and R. K.

Storms, Yeast 13, 151 (1997).

Copyright 2004, Elsevier Inc.All rights reserved.

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